Analysis of 67 RNA-seq datasets from various tissues at different stages of a model insect, Manduca sexta

Background: Manduca sexta is a large lepidopteran insect widely used as a model to study biochemistry of insect physiological processes. As a part of its genome project, over 50 cDNA libraries have been analyzed to profile gene expression in different tissues and life stages. While the RNA-seq data were used to study genes related to cuticle structure, chitin metabolism and immunity, a vast amount of the information has not yet been mined for understanding the basic molecular biology of this model insect. In fact, the basic features of these data, such as composition of the RNA-seq reads and lists of library-correlated genes, are unclear. From an extended view of all insects, clear-cut tempospatial expression data are rarely seen in the largest group of animals including Drosophila and mosquitoes, mainly due to their small sizes.Results: We obtained the transcriptome data, analyzed the raw reads in relation to the assembled genome, and generated heatmaps for clustered genes. Library characteristics (tissues, stages), number of mapped bases, and sequencing methods affected the observed percentages of genome transcription. While up to 40% of the reads were not mapped to the genome in the initial Cufflinks gene modeling, we identified the causes for the mapping failure and reduced the number of non-mappable reads to <8%. Similarities between libraries, measured based on library-correlated genes, clearly identified differences among tissues or life stages. We calculated gene expression levels, analyzed the most abundantly expressed genes in the libraries. Furthermore, we analyzed tissue-specific gene expression and identified 18 groups of genes with distinct expression patterns.Conclusion: We performed a thorough analysis of the 67 RNA-seq datasets to characterize new genomic features of M. sexta. Integrated knowledge of gene functions and expression features will facilitate future functional studies in this biochemical model insect.Peer reviewedBiochemistry and Molecular BiologyEntomology and Plant Patholog

Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation

Using BUSCO to Assess Insect Genomic Resources

The increasing affordability of sequencing technologies offers many new and exciting opportunities to address a diverse array of biological questions. This is evidenced in entomological research by numerous genomics and transcriptomics studies that attempt to decipher the often complex relationships amongst different species or orders and to build ‘omics’ resources to drive advancement of the molecular understanding of insect biology. Being able to gauge the quality of the sequencing data is of critical importance to understanding the potential limitations on the types of questions that these data can be reliably used to address. This chapter details the use of the Benchmarking Universal Single-Copy Orthologue (BUSCO) assessment tool to estimate the completeness of transcriptomes, genome assemblies, and annotated gene sets in terms of their expected gene content

Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera

Background: Rapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology. Results: Comparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects. Conclusions: Our results identify a burst of genetic novelty amongst sperm proteins that may be associated with the origin of heteromorphic spermatogenesis in ancestral Lepidoptera and/or the subsequent evolution of this system. This pattern of genomic diversification is distinct from the remainder of the genome and thus suggests that this transition has had a marked impact on lepidopteran genome evolution. The identification of abundant sperm proteins unique to Lepidoptera, including proteins distinct between specific lineages, will accelerate future functional studies aiming to understand the developmental origin of dichotomous spermatogenesis and the functional diversification of the fertilization incompetent apyrene sperm morph

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

<p>Abstract</p> <p>Background</p> <p>Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp <it>Chelonus inanitus </it>(Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences.</p> <p>Results</p> <p>About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein.</p> <p>An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the <it>Chelonus </it>lineage. Venom components specific to <it>C. inanitus </it>included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins.</p> <p>Conclusions</p> <p>The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of <it>C. inanitus </it>appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.</p

The Salivary Secretome of the Tsetse Fly Glossina pallidipes (Diptera: Glossinidae) Infected by Salivary Gland Hypertrophy Virus

Tsetse fly (Diptera; Glossinidae) transmits two devastating diseases to farmers (human African Trypanosomiasis; HAT) and their livestock (Animal African Trypanosomiasis; AAT) in 37 sub-Saharan African countries. During the rainy seasons, vast areas of fertile, arable land remain uncultivated as farmers flee their homes due to the presence of tsetse. Available drugs against trypanosomiasis are ineffective and difficult to administer. Control of the tsetse vector by Sterile Insect Technique (SIT) has been effective. This method involves repeated release of sterilized males into wild tsetse populations, which compete with wild type males for females. Upon mating, there is no offspring, leading to reduction in tsetse populations and thus relief from trypanosomiasis. The SIT method requires large-scale tsetse rearing to produce sterile males. However, tsetse colony productivity is hampered by infections with the salivary gland hypertrophy virus, which is transmitted via saliva as flies take blood meals during membrane feeding and often leads to colony collapse. Here, we investigated the salivary gland secretome proteins of virus-infected tsetse to broaden our understanding of virus infection, transmission and pathology. By this approach, we obtain insight in tsetse-hytrosavirus interactions and identified potential candidate proteins as targets for developing biotechnological strategies to control viral infections in tsetse colonies

A Deep Insight into the Sialotranscriptome of the Gulf Coast Tick, Amblyomma maculatum

Background: Saliva of blood sucking arthropods contains compounds that antagonize their hosts ’ hemostasis, which include platelet aggregation, vasoconstriction and blood clotting; saliva of these organisms also has anti-inflammatory and immunomodullatory properties. Perhaps because hosts mount an active immune response against these compounds, the diversity of these compounds is large even among related blood sucking species. Because of these properties, saliva helps blood feeding as well as help the establishment of pathogens that can be transmitted during blood feeding. Methodology/Principal Findings: We have obtained 1,626,969 reads by pyrosequencing a salivary gland cDNA library from adult females Amblyomma maculatum ticks at different times of feeding. Assembly of this data produced 72,441 sequences larger than 149 nucleotides from which 15,914 coding sequences were extracted. Of these, 5,353 had.75 % coverage to their best match in the non-redundant database from the National Center for Biotechnology information, allowing for the deposition of 4,850 sequences to GenBank. The annotated data sets are available as hyperlinked spreadsheets. Putative secreted proteins were classified in 133 families, most of which have no known function. Conclusions/Significance: This data set of proteins constitutes a mining platform for novel pharmacologically activ